The project aims to develop ratiometric near-infrared (NIR) fluorescent probes for the precise and quantitative analysis of lysosomal pH in living cells in living cells. These probes will have many advantages such as emission and absorption maxima in the NIR region around 650-800 nm for deep tissue penetration to avoid cell damage and interference from autofluorescence, large pseudo-Stokes shifts to overcome measurement errors by excitation and scattered lights, ratiometric responses with dual excitation and emission capability, excellent water solubility, high stability, high cell permeability, good biocompatibiliy, excellent intracellular retention, high selectivity, sensitivity and fast response to pH. We will design and prepare highly water-soluble FRET-based ratiometric near-infrared fluorescent probes for lysosomal pH in living cells by using near-infrared fluorescent dyes with the spirocyclic structure as acceptors and BODIPY dyes as donors. Large pseudo-Stokes shift of fluorescent probes will be achieved by using a short 1,2,3-triazole bridge between the acceptor and donor to accomplish efficient FRET from the BODIPY donor to the acceptor with the spirocyclic structure and overcome measurement errors by excitation and scattered lights with dual excitation or emission capability. High water solubility of the fluorescent probes will be achieved by introducing carbohydrate residues such as mannose and lactose with longer oligo(ethylene glycol) tethered spacers to the acceptor and BODIPY donors. There will be not any FRET between the donor and acceptor under physiological conditions (pH 7.4), and only emission of the BODIPY donors at 700 nm and 720 nm at excitation wavelength of 670 nm and 690 nm will be observed, respectively because the acceptor fluorescent dye will keep the closed form of the spirolactam ring at pH 7.4. A lysosomal acidic environment will trigger the opening of the acceptor spirolactam ring and significantly increase fluorescence intensity of the acceptor at 743 nm and 763 nm, and the intensity ratios (I743 nm/I690 nm and I763 nm/I720 nm) with excitation wavelengths of 670 nm and 690 nm via efficient FRET from the BOIDPY dye to the acceptor, respectively, which will provide the detection of pH changes in lysosome by both normal fluorescence and ratiometric fluorescence methods. We will apply these fluorescent probes for the precise and quantitative analysis of lysosomal pH changes in HEK293 cells, neuronal cells, and breast cancer cells to study cellular functions for insightful understanding of physiological and pathological processes.